Francesca Canyelles I Font, Krzysztof Żukowski, Masroor A Khan, Dorota Kwiatek, Jacek L Kolanowski
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引用次数: 0
Abstract
Bioluminescent high-throughput screening (HTS) assays, based largely on the activity of firefly (FLuc), Renilla (RLuc), and/or NanoLuc (NLuc) luciferases, are widely utilised in research and drug discovery. In this study, we quantify the luciferase-based real-life HTS assay interference from biologically and environmentally relevant metal ions ubiquitously present in buffers, environmental and biological matrices, and as contaminants in plastics and compound libraries. We also provide insights into the cross-effects of metal ions and other key experimental and biological reagents (e.g., buffer types, EDTA, and glutathione) to inform HTS assay design, validation, and data interpretation. A total of 21 ions were screened in three robust HTS assays ("SC" assays) based on the luminescence of FLuc, RLuc, and NLuc luciferases. Three newly optimised HEPES buffer variants ("H" assays) were developed for direct luciferase comparison. Interference in bioluminescent signal generation was quantified by calculating the IC50 values from concentration-dependent experiments for selected highly active and relevant metal ions. Metal ion inhibition mechanisms were probed by variations in specific reagents, EDTA, GSH, and the sequence of addition and buffer composition. In this study, we revealed a significant impact of metal ions' salts on luciferase-mediated bioluminescence, even at biologically and environmentally relevant concentrations. The extent of signal interference largely aligned with the Irving-Williams series of metal ion-ligand affinities (Cu > Zn > Fe > Mn > Ca > Mg), supporting previous reports on metal ion-dependent FLuc inhibition. However, the absolute magnitude and relative extent of signal reduction by metal ions' salts differed between SC and H assays and between luciferases, suggesting a complex network of metal ions' interactions with enzymes, substrates, reactants, and buffer elements. The diversity of the tested conditions and variability of responses provided insights into potential interference mechanisms and synergies that may exacerbate or alleviate interference. The beneficial influence of EDTA and the impact of glutathione, present natively in cells, on bioluminescence readout were pinpointed. Given the ubiquity of metal ions in analysed samples, the causative role in false-positive generation in drug discovery, and the wide breadth of luciferase-based assays used in screening, awareness and quantification of metal influence are crucial for developing assay validation protocols and ensuring reliable screening data, ultimately increasing the critical robustness of bioluminescence-based HTS assays.
期刊介绍：
Frontiers in Chemistry is a high visiblity and quality journal, publishing rigorously peer-reviewed research across the chemical sciences. Field Chief Editor Steve Suib at the University of Connecticut is supported by an outstanding Editorial Board of international researchers. This multidisciplinary open-access journal is at the forefront of disseminating and communicating scientific knowledge and impactful discoveries to academics, industry leaders and the public worldwide.
Chemistry is a branch of science that is linked to all other main fields of research. The omnipresence of Chemistry is apparent in our everyday lives from the electronic devices that we all use to communicate, to foods we eat, to our health and well-being, to the different forms of energy that we use. While there are many subtopics and specialties of Chemistry, the fundamental link in all these areas is how atoms, ions, and molecules come together and come apart in what some have come to call the “dance of life”.
All specialty sections of Frontiers in Chemistry are open-access with the goal of publishing outstanding research publications, review articles, commentaries, and ideas about various aspects of Chemistry. The past forms of publication often have specific subdisciplines, most commonly of analytical, inorganic, organic and physical chemistries, but these days those lines and boxes are quite blurry and the silos of those disciplines appear to be eroding. Chemistry is important to both fundamental and applied areas of research and manufacturing, and indeed the outlines of academic versus industrial research are also often artificial. Collaborative research across all specialty areas of Chemistry is highly encouraged and supported as we move forward. These are exciting times and the field of Chemistry is an important and significant contributor to our collective knowledge.
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